Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain

It is now well accepted that the structural protein Pr55Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development

Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice

<p>Abstract</p> <p>Background</p> <p>Among patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with <it>Pseudomonas aeruginosa </it>(<it>P. aeruginosa</it>). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17β-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with <it>P. aeruginosa </it>by a Th17-mediated mechanism.</p> <p>Results</p> <p>Exogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the <it>P. aeruginosa </it>strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against <it>P. aeruginosa </it>in vitro.</p> <p>Conclusions</p> <p>Our data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with <it>P. aeruginosa </it>in the CF lung.</p

A Connection between Colony Biomass and Death in Caribbean Reef-Building Corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994–2007), eleven years in the Exuma Cays, Bahamas (1995–2006), and four years in Puerto Morelos, Mexico (2003–2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1–4 m) compared to deeper-dwelling conspecifics (12–15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels

Genetic Traces of Recent Long-Distance Dispersal in a Predominantly Self-Recruiting Coral

Understanding of the magnitude and direction of the exchange of individuals among geographically separated subpopulations that comprise a metapopulation (connectivity) can lead to an improved ability to forecast how fast coral reef organisms are likely to recover from disturbance events that cause extensive mortality. Reef corals that brood their larvae internally and release mature larvae are believed to show little exchange of larvae over ecological times scales and are therefore expected to recover extremely slowly from large-scale perturbations.Using analysis of ten DNA microsatellite loci, we show that although Great Barrier Reef (GBR) populations of the brooding coral, Seriatopora hystrix, are mostly self-seeded and some populations are highly isolated, a considerable amount of sexual larvae (up to approximately 4%) has been exchanged among several reefs 10 s to 100 s km apart over the past few generations. Our results further indicate that S. hystrix is capable of producing asexual propagules with similar long-distance dispersal abilities (approximately 1.4% of the sampled colonies had a multilocus genotype that also occurred at another sampling location), which may aid in recovery from environmental disturbances.Patterns of connectivity in this and probably other GBR corals are complex and need to be resolved in greater detail through genetic characterisation of different cohorts and linkage of genetic data with fine-scale hydrodynamic models

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy

Mycobacterium abscessus-Induced Granuloma Formation Is Strictly Dependent on TNF Signaling and Neutrophil Trafficking

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections